UCSF

The ability to sense force is critical for virtually all systems of the human body, including the cardiovascular, respiratory, and digestive systems as well as the sensory systems of touch and hearing. The identity of the protein(s) converting physical stimuli to electrical impulse generation and cellular behavior is unknown in mammals. In order to identify molecules involved in mechanotransduction, it is necessary to develop high-throughput methods to assess the function of many genes at once. We have addressed this issue by employing a stretch stimulus in conjunction with real-time calcium imaging and are now able to view responses of hundreds of cells simultaneously. Using this system, we have uncovered the basic principles of stretch-activated channel activity in both sensory neurons of the trigeminal and dorsal root ganglia and in vascular smooth muscle cells. We find that in all of these cell types, a pulse of stretch results in a fast rise in intracellular calcium that decays exponentially after release of stretch. By removing calcium outside these cells, we demonstrate that responses in neurons and in smooth muscle cells are derived from extracellular calcium entry, implying the activity of stretch-activated ion channels. Pharmacological tools were used to further describe the characteristics of this calcium influx and to uncover differences between neuronal and vascular mechanotransduction.

Using our knowledge of response properties in these cells, we exploited the stretch technique to design two expression cloning screens for neuronal cDNAs

mediating stretch sensitivity. Furthermore, we screened novel spider, scorpion, and cone snail venoms for novel peptides that could inhibit stretch responses in smooth muscle cells. The ability to monitor many cells at once enables these and many other types of loss- and gain-of-function screens that were not possible in the past. This stretch assay will accelerate both the characterization of other mechanosensitive cell types and the discovery of molecules responsible for mechanotransduction in mammals.